Enterobacter sakazakii is a motile peritrichous, gram-negative rod from the family Enterobacteriaceae. This organism was previously known as a “yellow pigmented Enterobacter cloacae” until 1980, when it was introduced as a new species based on differences in DNA-DNA hybridization, biochemical reactions, and antibiotic susceptibility. E. sakazakii is a rare, but life-threatening cause of sepsis, necrotizing enterocolitis, and neonatal meningitis. In general, the reported case-fatality rate varies from 40-80% among newborns diagnosed with this type of severe infection.
Although the natural habitat of E. sakazakii is unknown, milk-based, powdered infant formula has been epidemiologically identified as the source of E. sakazakii infections, as was reported in an alert issued to United States Health Care Professionals by the United States Food and Drug Administration (USFDA) in April of 2002. In addition to powdered milk-based formulas, powdered human milk fortifiers may also pose a hazard.
Due to the extensive quantity and variety of bacteria present in the clinical environment, it is desirable to have methods for isolating and differentially identifying a single type of bacteria from a mixture of bacteria. A common approach to identifying bacteria is based on their appearance and/or growth characteristics in different types of culture media. To aid in bacterial isolation and identification, a growth medium may be both “selective” and “differential”. A selective medium is designed to suppress the growth of some microorganisms while allowing the growth of others (i.e., they select for certain microbes). A differential medium is designed to allow the growth of more than one microorganism of interest, but with visually or morphologically distinguishable colonies.
Several types of media, including Violet Red Bile Glucose agar (VRBG) and Tryptic Soy agar (TSA), have been used for the isolation and enumeration of E. sakazakii. Both of these types of media detect Enterobacteriaceae that produce characteristic yellow-pigmented colonies. However, these are generally not selective enough to reliably identify and distinguish E. sakazakii from other yellow pigment-producing Enterobacteriaceae present in clinical specimens or fresh water (Leclerc, H. et al. Annu. Rev. Microbiol. 55:201-234, 2001).
Other assays for E. sakazakii involve the use of the α-glucosidase substrate, 4-nitrophenyl-α-D-glucopyranoside. This approach has limitations because the yellow breakdown product, 4-nitrophenol, is easily diffusible on agar making it difficult to read (James, A L et al., Appl. Envirn. Microbiol. 62:3868-3870, 1996; Manafi, M., et al. Microbiological Reviews. 55:335-348, 1991). In addition, bacteria grown on TSA require a long (48-72 h) incubation time to produce yellow-pigmented colonies.
There is therefore, a pronounced need in the art for novel and improved methods for the isolation and detection of E. sakazakii that are more specific and rapid than currently-used methods.